Limits...
A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation.

Nakanishi S, Sanderson BW, Delventhal KM, Bradford WD, Staehling-Hampton K, Shilatifard A - Nat. Struct. Mol. Biol. (2008)

Bottom Line: We also identified several cis-regulatory residues on the histone H3 N-terminal tail, including histone H3 lysine 14 (H3K14), which are required for normal levels of H3K4 trimethylation.Several previously uncharacterized trans-regulatory residues on histones H2A and H2B form a patch on nucleosomes and are required for methylation mediated by COMPASS.This library will be a valuable tool for defining the role of histone residues in processes requiring chromatin.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA.

ABSTRACT
Methylation of histone 3 lysine 4 (H3K4) by yeast Set1-COMPASS requires prior monoubiquitination of histone H2B. To define whether other residues within the histones are also required for H3K4 methylation, we systematically generated a complete library of the alanine substitutions of all of the residues of the four core histones in Saccharomyces cerevisiae. From this study we discovered that 18 residues within the four histones are essential for viability on complete growth media. We also identified several cis-regulatory residues on the histone H3 N-terminal tail, including histone H3 lysine 14 (H3K14), which are required for normal levels of H3K4 trimethylation. Several previously uncharacterized trans-regulatory residues on histones H2A and H2B form a patch on nucleosomes and are required for methylation mediated by COMPASS. This library will be a valuable tool for defining the role of histone residues in processes requiring chromatin.

Show MeSH

Related in: MedlinePlus

Schematic representation of the experimental procedure. (a) Scanning histone mutagenesis with alanine (SHIMA). A library of alanine mutants at all residues of the four core histones, except the wild-type alanine residues in yeast S. cerevisiae, was systematically generated. (b) High-throughput yeast transformation and global proteomic screen (GPS) of S. cerevisiae histone mutants. The plasmids containing alanine point mutations within histone genes were generated by site-directed mutagenesis (Methods). The entire collection of histone alanine mutant libraries in S. cerevisiae was generated by transforming yeast shuffle strains (YBL 574 for H3 and H4, 4131 for H2A and H2B) and selection for the transformants, followed by a second selection on 0.1% (w/v) 5-FOA to remove wild-type histones. The collection of histone mutants was further analyzed by GPS to identify the amino acid residues required for proper H3 methylation.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2562305&req=5

Figure 1: Schematic representation of the experimental procedure. (a) Scanning histone mutagenesis with alanine (SHIMA). A library of alanine mutants at all residues of the four core histones, except the wild-type alanine residues in yeast S. cerevisiae, was systematically generated. (b) High-throughput yeast transformation and global proteomic screen (GPS) of S. cerevisiae histone mutants. The plasmids containing alanine point mutations within histone genes were generated by site-directed mutagenesis (Methods). The entire collection of histone alanine mutant libraries in S. cerevisiae was generated by transforming yeast shuffle strains (YBL 574 for H3 and H4, 4131 for H2A and H2B) and selection for the transformants, followed by a second selection on 0.1% (w/v) 5-FOA to remove wild-type histones. The collection of histone mutants was further analyzed by GPS to identify the amino acid residues required for proper H3 methylation.

Mentions: We have generated a complete library of alanine mutants at all residues of the four core histones, except at the naturally occurring alanine residues in yeast S. cerevisiae (Fig. 1a). We have named this library SHIMA. Plasmids containing alanine point mutations within the histone genes were generated by site-directed mutagenesis (Methods). Each plasmid was then sequenced for confirmation. The entire collection of the histone alanine mutant library in S. cerevisiae was generated by transforming the plasmids into yeast histone shuffle strains, either YBL574 containing hht2/hhf2 encoding H3 and H4, or Y131 (Osley’s lab) containing hta1/htb1, encoding H2A and H2B37. We carried out a strain selection for transformants, followed by a second single-colony selection on 5-fluoroorotic acid (5-FOA; Fig. 1b) to remove wild-type histone plasmids containing the URA gene. At this point, we identified the residues that are essential for cell survival under normal growth conditions (Fig. 1b). We carried out three additional selections by YPD-5-FOA to ensure the complete removal of wild-type histone plasmids before making the final stock of the library. Individual strains in the final yeast histone mutation library were sequenced for confirmation of the mutation and to determine the absence of the corresponding wild-type histone copy. The key for each plate is shown in Figure 2. This complete yeast mutation collection is now available to our colleagues.


A comprehensive library of histone mutants identifies nucleosomal residues required for H3K4 methylation.

Nakanishi S, Sanderson BW, Delventhal KM, Bradford WD, Staehling-Hampton K, Shilatifard A - Nat. Struct. Mol. Biol. (2008)

Schematic representation of the experimental procedure. (a) Scanning histone mutagenesis with alanine (SHIMA). A library of alanine mutants at all residues of the four core histones, except the wild-type alanine residues in yeast S. cerevisiae, was systematically generated. (b) High-throughput yeast transformation and global proteomic screen (GPS) of S. cerevisiae histone mutants. The plasmids containing alanine point mutations within histone genes were generated by site-directed mutagenesis (Methods). The entire collection of histone alanine mutant libraries in S. cerevisiae was generated by transforming yeast shuffle strains (YBL 574 for H3 and H4, 4131 for H2A and H2B) and selection for the transformants, followed by a second selection on 0.1% (w/v) 5-FOA to remove wild-type histones. The collection of histone mutants was further analyzed by GPS to identify the amino acid residues required for proper H3 methylation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2562305&req=5

Figure 1: Schematic representation of the experimental procedure. (a) Scanning histone mutagenesis with alanine (SHIMA). A library of alanine mutants at all residues of the four core histones, except the wild-type alanine residues in yeast S. cerevisiae, was systematically generated. (b) High-throughput yeast transformation and global proteomic screen (GPS) of S. cerevisiae histone mutants. The plasmids containing alanine point mutations within histone genes were generated by site-directed mutagenesis (Methods). The entire collection of histone alanine mutant libraries in S. cerevisiae was generated by transforming yeast shuffle strains (YBL 574 for H3 and H4, 4131 for H2A and H2B) and selection for the transformants, followed by a second selection on 0.1% (w/v) 5-FOA to remove wild-type histones. The collection of histone mutants was further analyzed by GPS to identify the amino acid residues required for proper H3 methylation.
Mentions: We have generated a complete library of alanine mutants at all residues of the four core histones, except at the naturally occurring alanine residues in yeast S. cerevisiae (Fig. 1a). We have named this library SHIMA. Plasmids containing alanine point mutations within the histone genes were generated by site-directed mutagenesis (Methods). Each plasmid was then sequenced for confirmation. The entire collection of the histone alanine mutant library in S. cerevisiae was generated by transforming the plasmids into yeast histone shuffle strains, either YBL574 containing hht2/hhf2 encoding H3 and H4, or Y131 (Osley’s lab) containing hta1/htb1, encoding H2A and H2B37. We carried out a strain selection for transformants, followed by a second single-colony selection on 5-fluoroorotic acid (5-FOA; Fig. 1b) to remove wild-type histone plasmids containing the URA gene. At this point, we identified the residues that are essential for cell survival under normal growth conditions (Fig. 1b). We carried out three additional selections by YPD-5-FOA to ensure the complete removal of wild-type histone plasmids before making the final stock of the library. Individual strains in the final yeast histone mutation library were sequenced for confirmation of the mutation and to determine the absence of the corresponding wild-type histone copy. The key for each plate is shown in Figure 2. This complete yeast mutation collection is now available to our colleagues.

Bottom Line: We also identified several cis-regulatory residues on the histone H3 N-terminal tail, including histone H3 lysine 14 (H3K14), which are required for normal levels of H3K4 trimethylation.Several previously uncharacterized trans-regulatory residues on histones H2A and H2B form a patch on nucleosomes and are required for methylation mediated by COMPASS.This library will be a valuable tool for defining the role of histone residues in processes requiring chromatin.

View Article: PubMed Central - PubMed

Affiliation: Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA.

ABSTRACT
Methylation of histone 3 lysine 4 (H3K4) by yeast Set1-COMPASS requires prior monoubiquitination of histone H2B. To define whether other residues within the histones are also required for H3K4 methylation, we systematically generated a complete library of the alanine substitutions of all of the residues of the four core histones in Saccharomyces cerevisiae. From this study we discovered that 18 residues within the four histones are essential for viability on complete growth media. We also identified several cis-regulatory residues on the histone H3 N-terminal tail, including histone H3 lysine 14 (H3K14), which are required for normal levels of H3K4 trimethylation. Several previously uncharacterized trans-regulatory residues on histones H2A and H2B form a patch on nucleosomes and are required for methylation mediated by COMPASS. This library will be a valuable tool for defining the role of histone residues in processes requiring chromatin.

Show MeSH
Related in: MedlinePlus